Rare diseases can provide critical insight into fundamental cellular mechanisms. One such disease, progressive osseous heteroplasia (POH), is a genetic disorder in which the body produces bone in skin, fat and skeletal muscle. Disorders of osteogenesis such as POH provide the opportunity to gain valuable information about the regulation of bone formation by revealing gene mutations that alter multi-potential precursor cells to promote osteoblast differentiation. We discovered that one of two copies of the GNAS gene is mutated in POH, establishing that inactivating GNAS mutations can alter cell fate to induce bone formation in inappropriate locations. The major product of the GNAS gene is Gs?, a G protein subunit that transmits signals from cell surface receptors to activate cAMP. Heterotopic (extra-skeletal) bone formation in POH patients often initiates within subcutaneous fat, suggesting common progenitor cells and a relationship, perhaps reciprocal, between osteogenesis and adipogenesis. Our investigations have shown that Gs? mRNA and protein expression and cAMP activity are reduced in cells from patients with POH and that decreased expression of GNAS and cAMP are associated with enhanced osteogenesis, while increased activation of cAMP signaling inhibits osteogenesis and favors adipogenesis. Our studies have demonstrated that GNAS plays a complex role in cell fate decisions and have directed us to further investigate the cellular and molecular mechanisms of osteogenic and adipogenic regulation by GNAS. In order to gain insight into this very complex process and based on our preliminary data, we will focus this proposal on investigations of cAMP signaling as a critical entry point to downstream signaling through GNAS/Gs? in regulating cell fate decisions. We hypothesize that cAMP signaling regulates early stage cell fate decisions that direct osteogenesis and adipogenesis. We propose three Specific Aims: Aim 1. Identify the roles and stages of GNAS and cAMP signaling in regulating cell fate decisions and tissue development using in vitro and in vivo assays. Aim 2. Examine interactions between cAMP and other signaling pathways in response to GNAS inactivation, focusing on cAMP crosstalk with the BMP pathway. Aim 3. Investigate the identity of progenitor cells that are recruited to form heterotopic bone and adipose tissue in response to GNAS inactivation through in vivo lineage tracing experiments. This proposal will provide new insights into the molecular pathways that regulate osteogenesis and adipogenesis under normal and pathological conditions, will delineate factors that control the differentiation of pluripotent stem cells in the musculoskeletal system into different lineages, and will provide an important foundation for the design of molecular diagnostic and treatment strategies for a wide range of human disorders of bone and adipose tissue. PUBLIC HEALTH RELEVANCE: Rare genetic disorders can provide valuable insight into fundamental cellular mechanisms that impact more common conditions. POH, a disease which forms extra-skeletal bone, is caused by gene mutations that regulate pluripotent stem cell differentiation in the musculoskeletal system. Our studies will investigate specific effects of the mutated POH gene on cell differentiation as well as identify progenitor cells and signaling pathways that regulate bone and fat formation. These studies will provide new insights leading to diagnostic and treatment strategies for a wide range of human disorders of bone and adipose tissue.